Production of riboflavin by ashbya gossyph with cobalt as additive



Patented Jan. 26, 1954 GOSSYPII WITH COBALT Richard J. Hickey,

AS ADDITIVE V Terre Haute, Ind., assignor to Commercial Solvents Corporation,

Terre Haute,

Ind.,-a corporation of Maryland N Drawing. Application August 5, 1949,

Serial No. 108,861

My invention relates to the production of riboe flavin and more specifically to a method for enhancing the yields of riboflavin produced by propagation of the organism Ashbya gossypiz' on nutrient media containing limited amounts of cobalt.

The production of riboflavin by the fermentation of nutrient mashes with the yeast Ashbya gossypz'i (also known as Nematospom gossymi) has been disclosed in U. S. Patent No. 2,445,128, granted July 13, 1948, to Fred W. Tanner, Jr. et al. According to this patent yields of 300 to 400 micrograms of riboflavin per ml. of culture liquor are produced by cultivating Ashbya gossypz'z' under aerobic conditions in a growth medium comprising 0.5 to 10.0% fermentable carbohydrates, a proteinaceous material, and a lipid. Examples of fermentable carbohydrates include glucose, sucrose, maltose and the like. Examples of proteinaceous materials include substances such as corn steep liquor, peptone, yeast and yeast products, distillers by-products, liver residues, animal tankage and other plant and animal proteinaceous substances some of which include the desired factors in bound form which factors must be released by treatment with acids, alkalis, enzymes, or combinations of such agents. Lipids such as corn oil and the like may be used.

In addition to the basic elements enumerated above, inorganic salts such as calcium carbonate, calcium chloride, monopotassium phosphate and trace elements such as copper, iron, manganese, and zinc are also usually added to the medium in small quantities unless already present in sufilcient quantities in one or more of the other ingredients of the medium.

The previously sterilized and inoculated medium is permitted to ferment at a temperature ranging from 15 to 40 C. and preferably between 24 and 30 C. while aerating the mash, preferably by introducing sterile air through spargers or other suitable devices located in the bottoms of the fermenters. The pH of the mash at the time of inoculation should'be within the range 4.0 and 8.0 and preferably between 5.0 and 7.5.

I have now discovered a means whereby the yields of riboflavin produced by the fermentations of nutrient media with the organism Ashbya gossypii under the conditions described above can be materially increased. I accomplish this by incorporating in the nutrient media of the type ordinarily fermentable by Ashbya gossyp'iz' limited amounts of cobalt. This is most 3 Claims. (Cl. 195-114) 2 conveniently accomplished by adding tolthe fere mentation medium a solution of colloidal cobalt or any water-soluble cobalt salt the anions of which are non-toxic to Ashbya, gossymi. Insole uble forms of cobalt can be used but less satisfactorily due to the difficulty of obtaining satisfactory dispersions of the small quantities of cobalt throughout the fermentation medium. Suitable forms of cobalt include such materials as the medium in the form of an aqueous solution so as to obtain more complete andmore rapid dispersion of the cobalt through the medium.

The amount of cobalt which I have found gives a stimulatory effect on the production of ribofiavin, when used in media for fermentation by Ashbya gossypii, in general ranges from 1 to 30 micrograms per milliliter of medium. The amounts required to give optimum results, however, will vary somewhat depending upon the character of fermentation medium employed, the length of the fermentation and other such factors. In some cases the production of riboflavin is inhibited instead of stimulated when in excess of 20 micrograms of cobalt per milliliter-"of medium are employed, whereas with other types of media, decided stimulatory eflects are programs of cobalt per milliliter.

The effect on riboflavin production of the incorporation of varying amounts of cobalt in fermentation media of various types is shown in the following examples.

EXAMPLE I A series of 1 liter flasks each containing milliliters of a fer lentation medium composed of 2% cerelose, 0.7% wheat fermentation slop was prepared and the pH adjusted to 6.8. Varying amountsof cobalt in the form of cobalt nitrate were added to these flasks, the contents sterilized and th mediumin each flask inoculated with about 1 ml. of a 48 were then incubated at 28 C. tation and aeration. The results obtained are shown inTable I.

peptone and 1% dried" 3 Table I each flask were inoculated with about 1 ml. of a culture of the organism Ashbya yossypii. The Riboflavin Produced flasks were then incubated at 28 C. for days Amt foob lt dd d micrograms/m1, mmmgmmS/ml with agitation and aeration. The results are d shown in Table IV. Nays 5 Table IV Riboflavin produced, micrograms ml. Amt. of Cobalt, micrograms/ml.

2 days 5 days Series 9 flasks containing 150 g 15 It isto be understood that any equivalents or still; megi emgmcrgngauclfiilg agygiige 21113311111231 5g changes whicoh WOllIddOCCIdlr to (line skillefidl m are co m ar are 0 e consi ere as yin W1 in e the following composition with the pH adjusted scope of my disclosure and mama to 6.5 was prepared: 2.5% cerelose, 0.5% corn 'Wha3 I claim is:

Steep liquor 025% beefsticki 025% amme 1. In a process for the production of riboflavin A (a commercial casein hydrolysate), 0.1% yeast by the propagation of the organism Ashbya extract. The medium in each flask was inoculated gossypii in nutrient media, the improvement g ggfi5gigggg223g; which comprises propagating said organism under ad b e e cultiv te d on the fermentation medium aeroblc condmons m a nutlilen-t medium contammg from 1 microgram to 20 micrograms of cobalt shwn Exafnple The g g per milliliter of medium in addition to any cobalt t at 28 g 7 z avl normally present in the constituents of the said aeration. The resu ts ob mm are s own in medium Table 30 2. In a process for the production of riboflavin Table II by the propagation of the organism Ashbya Rb fl d d gossypii in nutrient media, the improvement 5,555, 53, 3 which comprises propagating said organism under Amt. 0i Cobalt, micr grams/m aerobic conditions in a nutrient medium to which 7 days has been added from 1 to 20 micrograms of cobalt per milliliter of media in addition to any cobalt 560 580 normally present in the constituents of the said media.

3. In a process for the production of riboflavin by the propagation of the organism Ashbya gossypii in nutrient media comprising a fermentable carbohydrate and proteinaceous material the improvement which comprises propagat- 3 clays 5 days A series of flasks each containing 200 ml. of a fiermentamon me.d1um F 9 composl' ing said organism under aerobic conditions in tion, together with varying quantities of cobalt the presence of 1 to 20 micrograms of cobalt per in i g cobalt fia 2 5 milliliter of fermentation medium in addition to adlus ed 0 prepare cere any cobalt normally present in the constituents corn steep solids, 0.25% beef stick, 0.25% N-Z of the Said medium.

amine A (a commercial casein hydrolysate), and 0.1% yeast extract. After sterilization, the con- RICHARD HICKEY' tents of each flask were inoculated with about References Cited in t file of this patent 1 ml. of the culture of the organism Ashb'ya gossypii grown on a liquid medium containing UNITED STATES PATENTS corn steep liquor, N-Z amine A and beef stick. Number Name 7 Date The-flasks were then incubated at 28 C. for 7 2,326,425 Arzberger Aug. 10, 1943 days with agitation and aeration. The results 2,445,128 Tanner July 13,1943 are shown in Table III below. 2,595,499 Wood 'et al. May 6, 1952 Table 111 OTHER REFERENCES Riboflavin produced, Foster et al.: The Botanical Review, 'vol 4, micrograms/ml. April 1939; pages 207-11. Jacobs: The Chemistry and Technology of 3days 5days 7days Food and Food Products, 1944, vol 1, Interscienoe Pub. Inc.; pages 270-626. Book 95. 310 620 490 Porter: Bacterial Chemistry and Physiology, 10 ggg 1946, John Wiley & Sons, New York, pages 621, 15.- 230 580 570 627, 629.

' Wickersham et al.: Arch. of Biochem, 9, 1, EXAMPLE IV pages -98. A series of one liter flasks each containing Rickes 6t 1-! Sc ence 108, December 3, 1948, ml. of fermentation medium together with vary- 7 pages 634 35- mg amounts f cobalt in the form of cobalt Skmner: l-lenricis Molds, Yeasts and Actinonitrate was prepared having the following commycetesi 1947 John Wiley Sons, New York position with the pH adjusted to 6.8: 1.8% glupage 218. Book 530.

c o se, 0.7% peptone, and 1% dried wheat distil- Shine: Jour. Am. Chem. Soc. 70, pages 2614, lery slop. After sterilization, the contents of 75 2615. 

1. IN A PROCESS FOR THE PRODUCTION OF RIBOFLAVIN BY THE PROPAGATION OF THE ORGANISM ASHBYA GOSSYPII IN NUTRIENT MEDIA, THE IMPROVEMENT WHICH COMPRISES PROPAGATING SAID ORGANISM UNDER AEROBIC CONDITIONS IN A NUTRIENT MEDIUM CONTAINING FROM 1 MICROGRAM TO 20 MICROGRAMS OF COBALT PER MILLILITER OF MEDIUM IN ADDITION TO ANY COBALT NORMALLY PRESENT IN THE CONSTITUENTS OF THE SAID MEDIUM. 